Abstract Current routes for producing nanoscale devices require high-end nanofabrication techniques, such as focused ion beam milling or electron beam lithography coupled with the use of inorganic substrates. In spite for their unique operational characteristics, nanofluidic devices are difficult to be utilized for single-use applications as required for in vitro diagnostics. Novel fabrication strategies are conceived that will allow for the generation of nanofluidic devices made from thermoplastics using high-scale production modalities that yield devices at low- cost and with tight compliance, appropriate for single-use applications. The devices envisioned will employ single-molecule identification and/or quantification taking advantage of solid-phase molecular assays that can query for a variety of sequence variations in both DNA and RNA molecules using the same platform configuration. The fabrication strategy will employ high throughput nanoimprint lithography (NIL) used in combination with other micromachining techniques to produce mixed-scale structures. Utilizing an advanced assembly/bonding process specifically tailored for thermoplastic-based nanofluidic platforms, enclosed devices with high yield rates can be achieved. Using these nanofabrication techniques, a novel sensing platform will be explored that can take advantage of single-molecule digital counting to secure exquisite quantitative data that uses a non-optical readout modality. The sensor consists of a nanochannel flight tube with tapered 2D synthetic pores (opening <10 nm), which allows for identification of molecular entities via their characteristic flight time through a polymer nanochannel determined using transient current blockage events without the need to build in-plane and nano-gap electrodes. The sensor will also consist of microscale structures to allow for solid-phase molecular reactions that can generate unique molecular signatures of sequence variations in DNA and RNA that have been harvested from circulating markers such as biological cells, cell free DNA and exosomes. An in-depth understanding of single molecule behavior specific to polymer nanochannels and polymer solid-phase reactors is critical and will be extensively evaluated through experimentation and simulation. The sensor can be patterned over 4? wafers to provide the ability to do high throughput processing to search for rare molecular events and do so with high specificity and sensitivity. Wafer-scale production of the sensors will allow for using these compelling devices in a number of interesting biomedical applications such as searching for point mutations in genomic DNA isolated from circulating tumor cells, diagnosing stroke from exosomes through expression differences in their mRNA cargo or determining the methylation status of cell free DNA isolated from cancer patients.